Construction Of IKKα/β-shRNA Eukaryotic Expression Vectors And Establishment Of Stable Human Umbilical Vein Endothelial Cells Line (HUVECs) Expressing IKKβ Gene

Objective:This study is to construct a short hairpin RNA(shRNA) eukaryotic expression vector targeting the IKKα/βgenes by RNA interference(RNAi) and to establish stable Human Umbilical Vein Endothelial Cells line (HUVECs) expressing IKKβgene.Methods:The nucleotide sequences of IKKα/βgenes were retrieved from Genbank. According to the guidelines for shRNA design, four pairs of DNA sequence containing a small hairpin structure targeting the IKKα/βgenes were designed and synthesized. The synthesized sequences were cloned into pGPU6/GFP/Neo and identified by restriction enzyme analysis and sequencing analysis. After that, the recombinant vectors were transfected into HUVECs with lipofectamine 2000, and the most effective vectors were screened by assaying the IKKa/(3 protein levels. Finally, pGPU6/GFP/Neo-IKKβ-shRNA2 was transfected into HUVECs, then G418(600μg/ml) was added into the media changed every 3-5days. Western blot was used to identify the anti-G418 cell clones.Results:The restriction enzyme analysis and sequencing showed that the eight recombinant vectors were constructed exactly, termed as pGPU6/GFP/Neo-IKKa-shRNA1,2,3,4 and pGPU6/GFP/Neo-IKKβ-shRNA1,2,3,4. Western blot results showed that the expression levels of IKKa and IKKβproteins were upregulated, showing significant difference between HG group and Control group(P<0.01); the eukaryotic expression vectors containing the pGPU6/GFP/Neo-IKKα-shRNA4 and pGPU6/GFP/Neo-IKKβ-shRNA2 efficiently downregulated the protein expression levels of IKKa and IKKβgenes induced by elevated glucose concentration in HUVECs(P<0.01). Anti-G418 cell clones transfected with pGPU6/GFP/Neo-IKKβ-shRNA2 were formed via G418 resistance. Western blot results showed that the IKKβprotein expression of stable HUVECs expressing IKKβgene was significantly inhibited.Conclusion:The recombinant vectors are constructed successfully and can inhibit expression levels of IKKa and IKKβin HUVECs. Moreover, the stable HUVECs line expressing IKKβgene was established, which might be a beneficial tool for studying the insulin resistance in HUVECs.